Inhibitory NK cell receptors for MHC class I proteins, such as the Ly49 receptors in mice, are expressed by subsets of NK cells in an overlapping, predominantly monoallelic fashion. This 'variegated' gene expression pattern creates a repertoire of NK cell specificities for MHC molecules. The repertoire is additionally shaped by interactions with self-MHC class I molecules. The 'education' process has the effect of limiting the number of self-MHC-specific receptors expressed by each NK cell. Based on previously published and preliminary data, we have proposed that NK cells initiate expression of individual Ly49 genes in a stochastic, but nevertheless ordered and cumulative fashion. The initiation of new receptor gene expression during this process is, we propose, inhibited if previously expressed receptors interact strongly with self-MHC class I molecules. A detailed understanding of Ly49 gene regulation is clearly important for understanding the biological specificity and regulation of NK cells. Additionally, it is highly relevant for understanding the regulation of a growing number of other genes that reportedly exhibit variegated gene expression patterns, including cytokine genes and the transcription factor Pax5. Preliminary data provided in the application demonstrate that a genomic Ly49A transgene recapitulates the variegated gene expression pattern of the endogenous gene, and identifies a regulatory sequence in the gene ('HSS-1') that is necessary for transgene expression. We propose to experimentally address the molecular mechanisms that create a variegated pattern of Ly49 gene expression pattern, as well as cellular and molecular events that shape the NK repertoire. Specific Aim 1. To discriminate whether selection or another cellular mechanism shapes the Ly49 repertoire, and determine the role of the Shp-1 signaling pathway. Specific Aim 2. To determine whether DNA methylation is responsible for maintaining the variegated pattern of Ly49 gene expression. Specific Aim 3. To dissect the role of the novel regulatory element (HSS-1), and other regulatory sequences, in conferring a variegated gene expression pattern in transgenesis assays, and identify nuclear factors that interact with and regulate these elements. Specific Aim 4. To employ gene targeting to address the necessity of HSS-1 in Ly49A gene expression, and its role in controlling the developmental staging and extent of variegation of Ly49 gene expression.